Ocular therapy in keratoconjunctivitis sicca using topically applied androgens or TGF-β

ABSTRACT

The topical application, at a dose rate less than or equal to 0.1 mg day, to the ocular surface or adjacent regions of the eye of a preparation containing a therapeutically effective amount of an androgen or androgen analogue or a therapeutically effective amount of TGF-β is disclosed as a method of relieving the chronic and acute manifestation of dry eye signs and symptoms in keratoconjunctivitis sicca (KCS), for example in Sjogren&#39;s syndrome.

GOVERNMENT RIGHTS

Part of the work leading to this invention was made with United StatesGovernment funds under Grant No. EY05612 from the National Institutes ofHealth. Therefore, the U.S. Government has certain rights in thisinvention.

RELATED APPLICATIONS

This application is a continuation-in-part of Sullivan, U.S. patentapplication Ser. No. 08/971,768, filed Nov. 17, 1997, now U.S. Pat. No.5,958,912, which was a continuation-in-part of Sullivan, U.S. patentapplication Ser. No. 08/477,301, filed Jun. 7, 1995, now U.S. Pat. No.5,688,765, which was a continuation-in-part of Sullivan, U.S. patentapplication Ser. No. 08/124,842, filed Sep. 21, 1993, now U.S. Pat. No.5,620,921 (under reexamination as control no. 90/004,056), which was acontinuation under 37 CFR 1.62 of U.S. patent application Ser. No.07/871,657, filed Apr. 21, 1992, now abandoned, the whole of which arehereby incorporated by reference herein.

FIELD OF THE INVENTION

This invention relates to treating keratoconjunctivitis sicca (KCS),especially as manifested in Sjogren's syndrome.

BACKGROUND OF THE INVENTION

The preocular tear film plays an essential role in the maintenance ofcorneal integrity, the protection against microbial challenge and thepreservation of visual acuity (1).

These functions, in turn, are critically dependent upon the stability,tonicity and/or composition of the tear film structure, which includesan underlying mucin foundation (derived from conjunctival goblet cellsand conjunctival and corneal epithelial cells), a substantial, middleaqueous component (originating primarily from lacrimal gland acinar andductal epithelial cells) and an overlying lipid layer (secreted by themeibomian glands) (1,2). Alteration, deficiency or absence of the tearfilm may lead to intractable desiccation of the corneal epithelium,ulceration and perforation of the cornea, an increased incidence ofinfectious disease, and ultimately, severe visual impairment andblindness (2,3).

Throughout the world, countless individuals suffer from tear filmdysfunctions, which are collectively diagnosed as keratoconjunctivitissicca (KCS) or, simply, dry eye (1,2). These lacrimal abnormalities maybe subdivided into four general categories: (a) aqueous teardeficiencies, which are most frequently responsible for dry eye states,originate from lacrimal gland disorders and include autoimmune disease,congenital alacrima, paralytic hyposecretion or excretory ductobstruction; (b) mucin deficiency, which is observed in variousconjunctival cicatrization conditions, such as Stevens-Johnson syndrome,trachoma, pemphigoid, thermal and chemical burns, as well ashypovitaminosis A; (c) lipid abnormalities, which may occur duringmeibomian gland dysfunction (e.g., posterior blepharitis); and (d)diminished eyelid function (1).

By far, the greatest single cause of KCS worldwide, excluding thosecountries wherein trachoma remains epidemic, is Sjogren's syndrome (2).This syndrome, which is the second most common autoimmune disease(7,14), occurs almost exclusively in females and is characterized byinadequate mucin production, meibomian gland dysfunction, and aninsidious and progressive lymphocytic infiltration into the main andaccessory lacrimal glands, an immune-mediated, extensive destruction oflacrimal acinar and ductal tissues and the consequent development ofpersistent KCS (7-10). In primary Sjogren's syndrome, which afflictsabout 50% of the patient population, the disease is also associated withan immunological disruption of the salivary gland and pronouncedxerostomia. In secondary Sjogren's, the disorder is accompanied byanother disease, which is most often rheumatoid arthritis and lessfrequently systemic lupus erythematosus (SLE), scleroderma,polymyositis, polyarteritis nodosa, Hashimoto's thyroiditis, chronichepatobiliary disease, chronic pulmonary fibrosis, purpurahyperglobulinemia or Raynaud's phenomenon (2,11). During the course ofSjogren's syndrome, autoimmune sequelae may also encompass focallymphocytic adenitis of eccrine and mucosal glands, biliary cirrhosis,sclerosing cholangitis, pancreatitis, atrophic gastritis, interstitialnephritis and pneumonitis, peripheral vasculitis, B cell lymphoma and adiverse array of central and peripheral nervous system and skeletalmuscle complications (12,13).

The etiology of Sjogren's syndrome may be due to the interaction ofnumerous factors, including those of genetic, endocrine, neural, viraland environmental origin (15,16). However, a potential cause may relateto primary infection by, and reactivation of, Epstein-Barr virus (EBV)and/or cytomegalovirus (CMV) (17-20). These herpes viruses are presentin lacrimal and salivary glands of Sjogren's patients (17-20) and mayinduce the inappropriate HLA-DR expression, T helper/inducer cellactivation, B cell hyperactivity and autoantibody production evident inthese affected tissues (8). However, whether herpes, or even retroviral(21,22), action represents a cause of, or merely an epiphenomenon in,Sjogren's syndrome remains to be determined (23-25).

At present, a perception is that Sjogren's syndrome may be clinicallyirreversible (7), an autoimmune disease to be controlled, yet not cured(10). In the scientific literature, reports have suggested that systemicor topical administration of estrogens (4), cyclosporine A (6) orglucocorticoids (26) might alleviate the ocular manifestations of thisdisorder. However, other studies indicate that such pharmaceuticalexposures are ineffective (27-29) and, in fact, may accelerate and/oramplify the disease (28,30). Indeed, estrogen action may be involved inthe etiology of Sjogren's syndrome (30,31).

Others have suggested that tear stimulants, such as bromhexine (32) orisobutylmethylxanthine (33), might improve ocular symptoms. These drugeffects, though, may be subjective (34), susceptible to tachyphylaxis(4) and/or limited by the requirement for functional and responsivelacrimal tissue (4,35).

It has also been proposed that systemic androgen treatment might providea potential therapy for Sjogren's syndrome and its associated defects.This proposal is based upon the finding that autoimmune disorderscommonly display a sexual dichotomy, with estrogens increasing diseaseseverity in females and androgens suppressing autoimmune sequelae inmales (15,16,36-38). In fact, systemic androgen therapy has beenutilized to effectively diminish autoimmune expression in animals modelsof SLE, thyroiditis, polyarthritis and myasthenia gravis (15,38-43), aswell as the human condition of idiopathic thrombocytopenic purpura (44).However, research has also demonstrated that the systemic administrationof androgens to patients with primary or secondary Sjogren's syndrome orSLE is apparently unable to correct various peripheral manifestations ofthese disorders (49,54,55,62). In addition, systemic androgen treatmentof female patients with Sjogren's syndrome exposes these individuals topossible undesirable side effects, including virilization, menstrualirregularities (e.g., amenorrhea), hepatic dysfunction, edema,hematologic abnormalities, behavioral changes and metabolic alterations.Similarly, chronic treatment of males with systemic androgens has beencharacterized as dangerous (63), because of the numerous potential sideeffects. For these reasons, a recent report has indicated that systemicandrogen therapy is inappropriate for the treatment of the multipleimmune dysfunctions in Sjogren's syndrome (63).

Others have suggested that anti-viral compounds may represent a newtherapeutic approach for ocular disease in Sjogren's syndrome.Researchers have speculated that such compounds may be effective incounteracting the viral (e.g., EBV- and/or CMV)-induced infection inlacrimal tissue, that may possibly precipitate the gland'simmune-associated dysfunction (17,19,20). The potential efficacy of thisstrategy, though, is highly speculative: current scientific informationdoes not show definitively that these viruses are directly involved inthe pathogenesis or progression of Sjogren's syndrome (23-25).

Therefore, the currently prescribed, therapeutic approach for themanagement of KCS in Sjogren's syndrome is the frequent application ofartificial tear substitutes, which permit lubrication of the eye'santerior surface (3,4,5,9,10). Unfortunately, this therapy does notrepresent a cure and does not ameliorate the inherent, ocularimmunopathology and resulting KCS associated with this chronic,extremely uncomfortable and vision-threatening disease (3).

SUMMARY OF THE INVENTION

The invention generally features a new approach to the management ofKCS, especially as manifested in Sjogren's syndrome, the topicalapplication to the eye of a preparation containing a therapeutic amountof an androgen or androgen analogue, at a dose rate of less than 1mg/day, or a therapeutic amount of TGF-β. This method of treatment canalleviate the ocular manifestations of Sjogren's syndrome (e.g., as dueto lacrimal and meibomian gland dysfunction), the special symptoms thatcause great distress, while not exposing the patient to the possibleundesirable side effects of systemic treatment.

In one aspect, the invention features a method for treatingkeratoconjunctivitis sicca (KCS) that includes providing a therapeuticagent including a therapeutically effective amount of an androgen orandrogen analogue in a pharmaceutically acceptable substance, andadministering said therapeutic agent topically to the ocular surface orimmediate vicinity of an eye of a patient.

Preferably, the substance is phosphate buffered saline or a carriersubstance such as hyaluronate and the androgen or androgen compound hasunusual structural features; or the compound is a testosterone,4,5α-dihydrotestosterone, 17β-hydroxy-5α-androstane, or19-nortestosterone derivative; or the compound is a nitrogen-substitutedandrogen.

In another aspect, the invention features a similar treatment methodwherein the therapeutic agent includes TGF-β.

The invention also features measurement of the increased tear levels ofTGF-β to provide a diagnostic test to monitor the therapeutic effect oftopical treatment with androgen analogues.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Various androgen compounds, as herein discussed, significantly reducethe magnitude of lymphocyte infiltration in lacrimal tissue of animalmodels of Sjogren's syndrome. Androgens also stimulate the functionalactivity of both the lacrimal and meibomian glands. The nature ofandrogen action on autoimmune expression in lacrimal tissue appears tobe unique and lacrimal gland-specific. It also appears that this hormoneeffect is not linked to a generalized, systemic anti-inflammatoryfunction. Building on these new discoveries, the method of the inventioninvolves a rejection of the classical therapeutic approach to treatmentfor Sjogren's syndrome, a belief that any administered therapeutic agentmust be able to control all aspects of the disease. Because steroidhormones (e.g., glucocorticoids), with solubility characteristicsanalogous to those of androgens, rapidly gain access to adjacent oculartissues after topical application (78), it is proposed, instead, thattopical application of a therapeutic amount of an androgen or androgenanalogue to the eye be used to suppress lacrimal gland inflammation andto increase the functional activity of the lacrimal and meibomianglands, and thus treat the debilitating ocular manifestations of thisdisease. Topical application of a therapeutic androgen can provide forsymptomatic relief of the worst ocular symptoms of Sjogren's syndromewithout the chance of the patient experiencing the undesirable sideeffects of systemic administration. Furthermore, since theandrogen-induced suppression of lacrimal gland inflammation could bemediated through the induction of transforming growth factor-β (TGF-β),a potent immunosuppressive compound, local application of TGF-β shouldalso have the same effect.

During the past decade, it has become increasingly recognized that theendocrine system exerts a tremendous, regulatory impact on immunologicalexpression (15,16,36,37,45,64-76). The precise nature of this endocrinecontrol, though, appears to be both cell- and tissue-specific (45).Thus, depending upon the target, the consequence of hormone action maybe stimulation, antagonism or inhibition of immune function. Moreover,individual hormone effects on the immune system are often notgeneralized. Rather, endocrine influence may actually strengthen,diminish or elicit no effect on immunological activity in differenttissues (45). Given this background, it is not surprising that thesystemic administration of selected hormones (e.g., androgens) is unableto correct all immune defects in multidimensional, autoimmune disorders,such as Sjogren's syndrome or SLE.

Yet, if appropriate endocrine therapy could be targeted to specific,responsive tissues, hormone action could safely and effectivelyameliorate an immunopathology located in those particular tissues. Torelieve the symptoms that cause the most ocular distress in Sjogren'ssyndrome, those targeted, responsive tissues are the lacrimal andmeibomian glands. According to the invention, lipophilic, regulatoryhormones applied locally on or adjacent to the ocular surface can actdirectly on accessory and main lacrimal tissues and meibomian glands ofSjogren's syndrome patients and suppress the disease-related glandularinflammation and dysfunction in these tissues. This effect is completelyindependent of systemic hormone activity. The aim of thisimmunoendocrine interaction is to: (a) reduce lymphocyte infiltration inadjacent lacrimal tissue and thereby alleviate immune-mediateddestruction, and lymphocyte compression, of acinar and ductal cells; (b)permit accessory and/or palpebral lacrimal glands to secrete basal tearvolumes; (c) to enhance the function of meibomian glands and therebypromote increased stability and decreased evaporation of the tear film;and (d) avoid the side effects that parallel systemic exposure to thesehormones. In effect, topical androgen treatment can generate functionalregions of lacrimal tissue and augment meibomian gland activity, therebyenhancing tear output and maintenance and correcting the dry eyeproblem.

This pharmaceutical strategy has not been proposed previously. Mostprobably, this is because the mechanism of androgen action on immunefunction has been thought to be mediated through, or assisted by,factors from the thymus and hypothalamic-pituitary axis, or else involvedirect effects on lymphocytes (37,65,68,77). Specifically, androgenshave been thought to suppress immune processes indirectly by firstacting, for example, on the thymus, hypothalamic-pituitary axis, bonemarrow or spleen, which tissues would then release appropriate factorsor cells to mediate immunosuppression. Consequently, given thesepostulated mechanisms of androgen action, hormone administration to thesurface of the eye, prior to the method of the invention, would havebeen believed to be entirely ineffective: the quantity of steroiddelivered from the eye to the blood stream, and then to the thymus,hypothalamus, pituitary, bone marrow or spleen, would have been believedto be insufficient to induce the generation of enough immunosuppressiveagents to ameliorate lacrimal inflammation.

Topical Ocular Application of Androgens can Relieve the Immunopathologyof Sjogren's Syndrome

A critical requirement for the justification of topical ocularapplication therapy is to demonstrate that androgens suppress lacrimalgland immunopathology in Sjogren's syndrome. In addition, it isimportant to show that this androgen action is targeted to lacrimaltissue, and independent of generalized, systemic effects. In theexamples given below it is shown that all three of these criteria aremet, i.e., that androgens do suppress lacrimal gland immunopathology inSjogren's syndrome, that androgen action is targeted to lacrimal tissue,and that androgen action is independent of generalized, systemiceffects.

EXAMPLE I Androgen Influence on Lacrimal Gland Immunopathology in theMRL/Mp-lpr/lpr mouse model of Sjogren's Syndrome (47)

The purpose of the following study was to determine whether androgentherapy might inhibit the progression of, or reverse, autoimmune diseasein the lacrimal gland after the onset of Sjogren's syndrome. Towardsthat end, the study utilized adult, female MRL/Mp-lpr/lpr (MRL/lpr)mice, which are an animal model for both Sjogren's syndrome (50,51) andSLE (79,80). Lacrimal tissues of these mice, as in humans, containmultifocal and extensive lymphocytic infiltrates in perivascular andperiductal areas, significant glandular disruption and marked fibrosis(50,51).

Physiological or supraphysiological levels of testosterone wereadministered systemically, and not topically, because the location ofthe lacrimal gland in mice is inaccessible from the ocular surface. Theresults demonstrated that androgens exert a significant impact onautoimmune expression in lacrimal glands of MRL/lpr female mice.Administration of testosterone for 17 or 34 days dramatically reducedthe extent of lymphocyte infiltration in lacrimal tissue: this hormoneaction was time-dependent and involved marked diminutions in bothinfiltrate size and area. Moreover, hormone therapy appeared to reversethe inflammation-induced disruption of acinar and ductal epithelium. Ofinterest, there was no significant difference in experimental resultsbetween the physiological and supraphysiological doses of testosterone.In contrast, the magnitude of lymphocyte infiltration progressivelyincreased in lacrimal glands of placebo-treated mice during theexperimental time course. Testosterone therapy also significantlydiminished immunopathology in the submandibular gland, but the extent ofthis effect was less than found in lacrimal tissue.

EXAMPLE II Androgen Impact on Lacrimal Gland Immunopathology in theNZB/NZW F1 Mouse Model of Sjogren's syndrome (48)

The objective of this investigation was to assess the efficacy ofandrogen treatment for lacrimal disease by utilizing another autoimmune,animal model (NZB/NZW F1 [F1] mouse) of Sjogren's syndrome (52,59). Asin humans, lacrimal glands of this mouse strain, which displays afundamental B cell defect, harbor dense, lymphocytic aggregates (50,52),which contain a prevalence of B and helper T cells (58). Moreover, thismurine disease is accompanied by focal destruction of acinar and ductaltissue and apparent ocular surface dryness (50,52). In contrast, immunedysfunction in the MLR/lpr model appears to have a different etiologyand involves a basic, immunoregulatory disorder of T cells (47).

Autoimmune, female F1 mice were treated systemically with vehicle orvarying concentrations of testosterone for 0, 17, 34 or 51 days afterthe onset of disease; again, the systemic route for hormone treatmentwas utilized because lacrimal tissue in F1 mice may not be accessed fromthe ocular surface. Results showed that the extent of lymphocyteinfiltration increased dramatically in control mice during theexperimental time course. However, testosterone administration induced asignificant, time-dependent decrease in lymphocytic accumulation in thelacrimal gland. Following 34 to 51 days of androgen therapy, themagnitude of lymphocyte infiltration had been suppressed 22- to 46-fold,compared to that in placebo-treated tissue. This hormone effect wasassociated with significant reductions in the number of focalinfiltrates, the area of individual foci and the total quantity oflymphocyte infiltration per lacrimal section. In certain groups,testosterone exposure also stimulated a rise in tear volumes, relativeto those measured in the same mice prior to treatment. With fewexceptions, the impact of physiological and supraphysiologicaltestosterone treatment on lacrimal autoimmune expression in F1 mice wasessentially identical, the suppression of autoimmune disease.

EXAMPLE III Effect of Androgen Therapy in Sjogren's Syndrome: HormonalInfluence on Lymphocyte Populations and Ia Expressionin Lacrimal Glandsof MRL/lpr Mice

Previous research demonstrated that androgen treatment dramaticallycurtails lymphocyte infiltration in lacrimal glands of mouse models ofSjogren's syndrome. The purpose of this study was to determine whetherthis androgen action involves the selective suppression of specificlymphocyte populations or Class II antigen (i.e., Ia) expression inlacrimal tissue. Towards this end, autoimmune female MRL/Mp-lpr/lpr micewere administered placebo- or testosterone-containing compoundssystemically for 0, 17 or 34 days after the onset of disease. Resultsshowed that androgen exposure exerts both a quantitative and aqualitative influence on inflammatory cell populations in the lacrimalgland of MRL/lpr mice. Thus, testosterone, but not placebo, treatmentinduced a precipitous decrease in the total number of T cells, helper Tcells, suppressor/cytotoxic T cells, Ia-positive lymphocytes and Bcells. Androgen administration also significantly diminished thelacrimal density, as well as the frequency, of B220⁺ (i.e., possiblyimmature T) cells.

These findings, when compared with other observations (45,47,48,56),suggest that testosterone's anti-inflammatory activity may be unique andlacrimal gland-specific. First, the androgen-induced immunosuppressionin lacrimal tissue does not extend to peripheral lymph nodes (56,57),indicating that this steroid hormone does not cause a generalizeddepression in lymphocyte migration to, or proliferation in, systemic ormucosal sites. Second, testosterone exposure reduces the magnitude oflymphocytic infiltration in submandibular glands of MRL/lpr mice (47),but the nature of this hormonal influence may be unlike that found inlacrimal tissue, and the overall susceptibility of salivary focalinfiltrates to androgens and pharmacological agents appears quitedifferent from that found in lacrimal tissue (47). Third, androgensexert significant control over immunological functions in lacrimalglands, but not necessarily those of salivary or systemic tissues (45).

EXAMPLE IV Impact of Steroids and Immunosuppressive Agents on LacrimalAutoimmune Disease in the MRL/lpr Mouse Model of Sjogren's Syndrome

The objective of the following experiments was to determine whetherother steroid hormones or immunosuppressive agents might duplicate theeffect of testosterone on lacrimal gland autoimmunity. Female MRL/lprmice were treated with systemic vehicle, steroids or immunosuppressivecompounds for 21 days after disease onset. The pharmaceutical agentsevaluated in this study included: (a) testosterone, which, has beenshown to significantly reduce lacrimal gland inflammation; (b)19-nortestosterone, an anabolic androgen with attenuated virilizingactivity; (c) danazol, a synthetic steroid, which is utilized in thetreatment of certain immune diseases in humans (81) and is known todiminish specific, peripheral immune defects in MRL/lpr mice (60); (d)17β-estradiol, a female sex steroid, which has been hypothesized as apotential treatment for ocular disease in Sjogren's syndrome (4); (e) asynthetic, non-androgenic steroid, which apparently suppresseslymphocyte infiltration in salivary glands of F1 mice and corrects othersystemic autoimmune defects; (f) cyclosporine A, an anti-inflammatoryagent, which ameliorates specific, peripheral immune dysfunctions inMRL/lpr mice (61) and has been proposed as an effective therapeuticagent for lacrimal disease and KCS in Sjogren's syndrome (6); (g)dexamethasone, a potent anti-inflammatory glucocorticoid, that has beensuggested as a possible therapeutic agent for lacrimal immunopathologyin Sjogren's syndrome (26); and (h) cyclophosphamide, animmunosuppressive agent, that decreases various autoimmune sequelae insystemic (83-85) and salivary (59,82) sites in MRL/lpr mice. Thecomparative results demonstrated that the suppressive influence oftestosterone on focal infiltrate area, number of foci and percentagelymphocyte infiltration in lacrimal tissue was duplicated by theadministration of the anabolic androgen, 19-nortestosterone, orcyclophosphamide, but not by therapy with estradiol, danazol, thesynthetic non-androgenic steroid, cyclosporine A, or dexamethasone. Inaddition, testosterone, 19-nortestosterone and cyclophosphamide, as wellas dexamethasone, reduced lymphocyte infiltration in the submandibulargland. However, neither androgen interfered with the pronouncedinflammation of lymphatic tissues, including the spleen, and superiorcervical and mesenteric lymph nodes. Androgen treatment alone alsostimulated an increase in the lacrimal gland output of total protein andIgA antibodies into tears; these antibodies, which protect the ocularsurface against bacterial colonization, viral attachment, parasiticinfestation and fungal- or toxin-induced impairment (46), are typicallydiminished in mucosal sites in Sjogren's syndrome (53).

Overall, these combined findings demonstrate that androgens, or theiranabolic analogues, suppress autoimmune expression in, and enhancetissue function of, lacrimal glands of animal models of Sjogren'ssyndrome. Androgen action also appears to represent a tissue-specificresponse independent of generalized, systemic effects, thus justifyingtopical ocular therapeutic application. Cyclophosphamide, the onlynon-androgen to reduce lymphocyte infiltration in lacrimal tissue uponsystemic administration, is not believed to be appropriate for topicaltherapy in humans because of its mode of action. This alkylating agent,which is thought to suppress autoimmune function by a directmodification of cellular DNA, must first be metabolized by the liverbefore becoming active. Therefore, cyclophosphamide would not be capableof local action upon topical application.

Local Ocular Application of TGF-β can Relieve the Immunopathology ofSjogren's Syndrome

The androgen-induced suppression of immunopathological lesions inautoimmune lacrimal tissue, and the parallel improvement in glandularfunction, could be mediated through an androgen interaction withepithelial cells, which would then cause the altered expression and/oractivity of epithelial cytokines in the lacrimal gland. If correct, thishypothesis would predict that: (a) epithelial cells are the target cellsfor androgen action in the lacrimal gland, and that androgen receptorsare located within epithelial, but not other (e.g., lymphocytic), cellsin lacrimal tissue; and (b) androgens increase the expression ofanti-inflammatory cytokines, or decrease the activity ofpro-inflammatory peptides, in the lacrimal gland. Therefore, to addressthis hypothesis, experiments were conducted to identify the presence,location and/or cellular distribution of androgen receptor protein andmRNA in lacrimal glands of various species and autoimmune, femaleMRL/lpr mice, as well as examine the impact of androgens on cytokinelevels in lacrimal tissue.

EXAMPLE V Presence, Location and Hormonal Regulation of AndrogenReceptors in Lacrimal Tissue

The mechanism(s) by which androgens regulate the immune expression inthe lacrimal gland undoubtedly involves an initial hormone associationwith specific androgen receptors. Androgen receptors appear to mediatealmost all known activities of androgens and are members of thesteroid/thyroid hormone/retinoic acid family of ligand-activatedtranscription factors (90-96). The location of androgen receptors inother tissues is predominantly intranuclear (97-99), due to the presenceof a nuclear targeting signal, homologous to that of the SV 40 large Tantigen, which occurs in the receptor hinge region immediately followingthe DNA-binding domain (93). Following androgen binding to the receptor,the monomeric, activated hormone-receptor complex associates with anandrogen response element in the control region of specific targetgenes, typically dimerizes with another androgen-bound complex and, incombination with appropriate silencers, tissue-specific and basalpromoter elements, regulates gene transcription (90,91,93,94,96). Thisandrogen activity results in the alteration of mRNA production andultimately protein synthesis in a variety of tissues (100-103); suchregulation of protein elaboration appears to be the primary action ofandrogens (90,104).

A critical prerequisite, though, for the androgen-induced regulation oftarget gene transcription is the presence and location of androgenreceptors in a given tissue (90). In fact, considerable research hasdemonstrated that a tissue's physiological responsiveness to steroids(e.g., androgens) is most often directly proportional to theconcentration of that steroid's receptor protein, as well as to theamount of its associated mRNA (90,105,106).

Therefore, to determine whether high affinity and specific androgenreceptors are present in the lacrimal gland, equilibrium binding methodswere employed with various tritiated steroids and with lacrimal tissuecytosol from young adult, orchiectomized or ovariectomized rats.Analysis revealed that a single class of saturable, high-affinity andspecific binding sites exist for androgens in lacrimal tissues of bothmale and female rats. To extend these findings, experiments were alsoperformed to evaluate whether high-affinity androgen binding sites arelocated specifically within acinar epithelial cells of the rat lacrimalgland. Towards that end, acinar epithelial cells were isolated fromlacrimal tissues of orchiectomized rats, processed for the preparationof cytosol and examined for tritiated dihydro-testosterone (DHT) bindingsites. This analysis identified the existence of high-affinity androgenreceptors within these cells.

To determine whether androgen receptor mRNA is present in lacrimaltissues of various species, the following studies were conducted. RNAwas isolated from lacrimal glands of male and/or female mice, rats,hamsters, guinea pig, rabbits and human, as well as from rat spleen(negative control), rat prostate and human prostate LNCaP cells(positive controls), then reverse transcribed into cDNA, amplified withspecific primers by polymerase chain reaction (PCR) and processed forSouthern blot hybridization with a 32P-labeled fragment of rat androgenreceptor cDNA. The size of amplified cDNA products was calculated bycomparison to a series of molecular weight standards run in adjacentlanes in the 1.5% agarose gel. The results showed that lacrimal glandsfrom all tested species contained a single, ˜1,273 bp band of androgenreceptor mRNA, which was identical to that observed in rat prostatictissue and human prostatic LNCaP cells. In addition, the same RT-PCRresults were found if "human-specific" oligomeric primers were utilizedto amplify mRNA from human lacrimal tissue, LNCaP cells and ratprostate.

For comparative purposes, studies were also conducted to examine humanand/or rat lacrimal glands for the existence of androgen receptor mRNAby the use of ribonuclease protection assays, as well as Northern blottechniques. In the former experiments, total cellular RNA was isolatedfrom human lacrimal gland autopsy specimens (n=5 males, 1 female), LNCaPcells, as well as rat lacrimal, prostatic and splenic tissues, thenhybridized to 32P-labeled, human or rat androgen receptor riboprobes.Findings showed that androgen receptor mRNA occurred in both human andrat lacrimal glands, and that the size was equivalent to that of theprostatic androgen receptor mRNA. In the ribonuclease protection assayswith rat tissues, no androgen receptor mRNA was detected in the spleen,whereas G-3-PDH mRNA was evident in all rat lacrimal, prostatic andsplenic samples. For comparison, it has also been demonstrated thatandrogen receptor mRNA occurs in rat lacrimal tissue, and that themolecular size (˜10 kb; by Northern blots) is identical to that observedin the prostate, an androgen target organ (107).

To explore the endocrine basis for the androgen regulation of both thestructure and function of the lacrimal gland in a variety of species,studies were also designed to: (a) determine the cellular distributionof androgen receptors in the lacrimal gland; and (b) examine theinfluence of gender and the endocrine environment on the glandularcontent of these binding sites. Lacrimal glands were obtained fromintact, castrated, or sham-operated male or female adult rats, mice orhamsters, as well as from orchiectomized rats exposed to placebocompounds or physiological levels of testosterone. The cellular locationof androgen receptors was evaluated by utilizing an immunoperoxidaseprotocol, in which a purified rabbit polyclonal antibody to the ratandrogen receptor was used as the first antibody. Findings with lacrimalglands showed that: (a) androgen receptors are located almostexclusively in nuclei of epithelial cells; (b) the cellular distributionor intranuclear density of these binding sites is far more extensive inglands of males, as compared to females; (c) orchiectomy, but notsham-surgery, leads to a dramatic reduction in the immunocytochemicalexpression of androgen receptors; and (d) testosterone administration toorchiectomized rats induces a marked increase in androgen receptorcontent, relative to that in placebo-exposed glands. Overall, thesefindings demonstrate that gender and the endocrine system maysignificantly influence the distribution of androgen binding sites inrat lacrimal tissue. Moreover, these results show that androgensup-regulate their own lacrimal gland receptors.

To assess whether the androgen control of, and gender-relateddifferences in, androgen receptor protein in the lacrimal gland areassociated with variations in the expression of androgen receptor mRNA,the following study was performed. Orchiectomized and ovariectomizedSprague-Dawley rats were administered subcutaneous implants of placebo-or testosterone-containing pellets for 7 days. Lacrimal glands wereobtained from these animals, as well as intact male, female andsham-operated rats, and then processed for the measurement of androgenreceptor mRNA by semi-quantitative reverse transcription (RT)-PCR. Allandrogen receptor mRNA determinations were standardized to the β-actincontent in the same RNA sample. The results of this study showed thatthe levels of androgen receptor mRNA are significantly higher inlacrimal tissues of females, as compared to males. In addition, thefindings demonstrated that orchiectomy increases, and androgen treatmentreduces, androgen receptor mRNA content in lacrimal tissue. Thus, theeffects of gender and androgen exposure on AR mRNA expression in thelacrimal gland are the opposite to those observed with AR protein. Thistype of AR autoregulation has also been found by other investigators incertain reproductive tissues.

The above findings demonstrate that androgen receptor protein and mRNAare present in lacrimal tissues of a number of species and that thesebinding sites possess high affinity, are specific for androgens, and arelocated almost entirely within nuclei of epithelial cells. To extendthese results further, additional studies were performed to identify thecellular target(s) within autoimmune lacrimal tissue that may mediatethe immunosuppressive effect of androgens. In addition, the endocrineregulation of androgen receptors in these autoimmune lacrimal glands wasexplored. Adult, female MRL/lpr mice were exposed systemically tovehicle, steroid hormones or immunosuppressive agents for varying timeintervals after the onset of disease. Immediately before or aftertreatment, lacrimal glands were obtained and processed to determine thecellular distribution and nuclear density of androgen receptors byimmunoperoxidase and image analysis techniques, and to assess the levelsof androgen receptor mRNA. The findings demonstrated that: (a) androgenreceptors exist almost exclusively within nuclei of acinar and ductalepithelial cells in lacrimal tissue of MRL/lpr mice; (b) androgenreceptors are not detectable in the extensive lymphocytic populationsthat infiltrate the gland; (c) testosterone administration induces asignificant increase in the number of androgen receptor-containing cellsin, as well as the density of androgen receptors in epithelial cellnuclei of, lacrimal tissue; (d) hormone action is steroid-specific:administration of androgen analogues, but not estrogens, glucocorticoidsor cyclophosphamide, stimulate the accumulation of androgen receptors;(e) androgens autoregulate the amount of their receptor mRNA; and (f)androgen receptor density is significantly reduced following thewithdrawal of androgen therapy. These results show that epithelialcells, but not lymphocytes, are the androgen target cells in lacrimaltissue, and appear to mediate the androgen-related immunosuppression andfunctional enhancement in lacrimal glands of autoimmune female mice.These findings also demonstrate that androgens increase the expressionof their own receptor protein, and decrease the content of theirreceptor mRNA, in MRL/lpr lacrimal tissue.

In summary, these results show that epithelial cells, but notlymphocytes, are the androgen target cells in autoimmune lacrimaltissues, and that androgens up-regulate the expression of their ownreceptor protein in these cells. In addition, it has been shown that:(a) specific, high-affinity and saturable androgen binding sites existin rat lacrimal tissue; (b) the location of these receptors is almostexclusively within epithelial cells of lacrimal glands in non-autoimmunemice, rats and hamsters; (c) androgen receptor mRNA is present inlacrimal tissues of humans and numerous other species; (d) theappearance of androgen receptor protein and mRNA in the lacrimal glandis significantly influenced by gender and androgens; and (e) androgenact directly on epithelial cells of the rat lacrimal gland and thesehormone actions may be inhibited by cellular exposure to androgenreceptor, transcription and translation antagonists (108,109).

EXAMPLE VI Role of Epithelial Cell TGF-β in Lacrimal Gland AutoimmuneDisease

Autoimmune diseases invariably involve a deficiency in self tolerance,the generation of autoreactive immune cells, the activation ofproto-oncogenes and the expression of immune response genes to thedetriment of the host (110-112). However, an additional and extremelyimportant feature of autoimmune disorders is the inappropriate secretionof cytokines (111,112). These peptides, which are produced by a widevariety of cells, e.g., immune, epithelial, endothelial, and neural(112-116), have been termed the "hormones of the immune system" (117)and normally play an integral role in immunological defense (112,118).An imbalanced production and release of cytokines, though, may lead tothe subversion of tolerance to specific antigens, activation of effectorfunctions of T and B cells, stimulation of proto-oncogene, Class IIantigen and intercellular adhesion molecule expression, promotion of theinflammatory process and destruction of target cells, e.g., epithelial(110,112,117-120). Given these actions, cytokines have also beenimplicated as the "mediators of autoimmune disease" (121). At present,numerous cytokines appear to be involved in autoimmune disorders(111,112,117-119,122) and to contribute significantly to the etiologyand/or generation of inflammatory eye diseases (123,124).

Recently, studies have also implicated cytokines in the development andperpetuation of severe immunopathological lesions in exocrine tissues ofSjogren's syndrome patients (125), as well as in the striking decreasein glandular secretion that occurs in this disorder (126). Thus,salivary glands of individuals with Sjogren's syndrome show a tremendousincrease in the interleukin-1 (IL-1), IL-6 and tumor necrosis factor-α(TNF-α) mRNA levels in acinar epithelial cells, a substantial rise inIL-2, IL-10 and IFN-mRNA content in CD4 T cells, and a significantelevation in IL-1, IL-6, TNF-α, IL-10 and IFN-concentrations in saliva,relative to those amounts in healthy controls (127). Analyses of totalsalivary gland biopsies have also documented alterations in the mRNAexpression of these cytokines, as well as of IL-1β and TGF-β mRNA(128-130). With regard to lacrimal glands, a limited evaluation ofbiopsies from Sjogren's syndrome patients by RT-PCR has identifiedelevated amounts of IL-1β, IL-6 and IFN-mRNA (128,131). Of interest,these changes in cytokine circuitry might explain not only theinflammatory progression of this disease, but also the associatedxerophthalmia and xerostomia in Sjogren's syndrome. Thus,cytokine-induced lymphocyte infiltration may result in a significantdecline in the neural innervation of inflamed tissue (132). Moreover,certain cytokines (e.g., IL-1β) may directly suppress transmitterrelease by adrenergic and cholinergic nerves (133), thereby interferingwith the neural control of epithelial cell fluid secretion (126).

This aberrant expression of cytokines in Sjogren's syndrome may well bedue, in part, to the influence of sex steroids. In support of thishypothesis, estrogens, which may be involved in the pathogenesis,acceleration and amplification of Sjogren's syndrome (30,31), are knownto enhance the production of pro-inflammatory cytokines, e.g., IL-1 andTNF-α (134). Conversely, androgens, which decrease the manifestations ofmany autoimmune disorders (16,37,135), may elicit the generation ofimmunosuppressive cytokines (136). In fact, the sex steroid regulationof cytokine synthesis is believed to account for the distinct sexualdimorphism found in the incidence of autoimmune disease (122).

Indeed, the principle mechanism by which androgens suppress autoimmunedisease in lacrimal glands of female mouse models of Sjogren's syndromemay well be through the control of epithelial cell cytokine production.In support of this hypothesis, as has been described above, theanti-inflammatory action of androgens in lacrimal tissue appears to bemediated not through lymphocytes, but rather through epithelial cells.Moreover, epithelial cells in other tissues are known to secretenumerous cytokines, e.g., TGF-β (137), and also serve in exocrine sitesas active cellular participants in the glandular inflammation inSjogren's syndrome (138). In addition, as will be described below,androgens increase the mRNA and protein levels of the immunosuppressivecytokine, TGF-β1, in the lacrimal gland. This cytokine is thought toplay a protective role in Sjogren's syndrome, and increased expressionof TGF-β mRNA has been correlated with reduced inflammation in salivaryglands of Sjogren's syndrome patients (129). In contrast, the absence ofTGF-β1 leads to a pronounced lymphocytic infiltration into both lacrimaland salivary glands (139).

Therefore, to test this hypothesis, studies were performed identifyepithelial cell cytokines that may mediate, or be involved, in theandrogen-induced suppression of lacrimal gland inflammation in Sjogren'ssyndrome. An initial inquiry was whether lacrimal gland epithelial cellsexpress cytokines, that might be involved in the endocrine regulation ofimmune function in this tissue. These studies, which were conducted withhigh stringency, RT-PCR procedures, demonstrated that TGF-β1, TGF-β2,TGF-β3, IL-6, TNF-α and IL-1α mRNA may be detected consistently inlacrimal glands, as well as in isolated lacrimal acinar epithelialcells, of male and female rats. As a corollary to these studies, whetherlacrimal glands of autoimmune mice contain mRNAs for anti-inflammatory,as well as pro-inflammatory, cytokines was also examined. This research,which was performed with high stringency RT-PCR techniques, showed thatmRNA for IL-1α, IL-1β, IL-2 receptor, IL-6, TGF-β and TNF-α may bedetected consistently in lacrimal glands of autoimmune female MRL/lprmice. The identity of these amplified products was verified by agarosegel electrophoresis, molecular weight determinations and comparison toseveral positive controls (e.g., cDNA from MRL/lpr splenic tissue,P388D1 macrophages, and Clontech kit controls).

Given these findings, the next set of studies was designed to determinewhether the mRNA and protein levels of the immunoinhibitory cytokine,TGF-β1, in the lacrimal gland might be controlled by androgens, andinfluenced by gender. In the first series of experiments, total RNA wasisolated from lacrimal glands of intact, castrated or sham operated maleand female rats, as well as placebo- or testosterone-treatedorchiectomized rats. Following this isolation, TGF-β1 and β-actin mRNAwere analyzed by agarose gel electrophoresis, RT-PCR, Southernhybridization, autoradiography and densitometry. For control purposes,all TGF-β1 measurements were standardized to those of β-actin. Theresults demonstrated that: (a) levels of TGF-β1 mRNA were significantlyhigher in lacrimal tissues of intact male, as compared to those offemale, rats; (b) orchiectomy either decreased, or had no impact, onTGF-β1 mRNA content in lacrimal glands, whereas ovariectomy consistentlyhad no effect on TGF-β1 mRNA amounts; and (c) androgen treatmentsignificantly increased TGF-β1 mRNA expression in lacrimal tissues oforchiectomized rats, relative to that in glands of placebo-treatedcontrols. These data demonstrated that gender influences, and androgensregulate, TGF-β1 mRNA levels in the lacrimal gland.

A second series of studies examined whether androgen exposure mightsignificantly increase the content of TGF-β1 protein in lacrimal tissuesof autoimmune mice. Accordingly, lacrimal tissues were obtained fromfemale MRL/lpr mice after the onset of disease and following placebo,testosterone or cyclophosphamide treatment. Glands were then processedfor the acid extraction of proteins and the analysis of TGF-β1 levels byuse of a commercial assay. The results showed that androgen, but notcyclophosphamide, administration stimulated a significant rise in thetotal amount of TGF-β1 protein in lacrimal tissues of female MRL/lprmice, as compared to levels in tissues of placebo controls.

Overall, these findings show that the mRNAs for cytokines believed toplay a major role in exocrine tissue inflammation in Sjogren's syndromeare present in lacrimal tissues of normal rats and autoimmune mice. Inaddition, these results demonstrate that androgens stimulate theaccumulation of TGF-β1 mRNA and protein in the lacrimal gland. TGF-β1,in turn, is known to exert profound immunosuppressive activity,including the inhibition of T and B cell proliferation, cytotoxic T cellgeneration, natural and lymphokine-activated killing, T cell adhesion tothe endothelium, macrophage function and IL-1, TNF and IFN-γ production,and is believed to down regulate inflammation in exocrine glands inSjogren's syndrome (129,139,140). Consequently, the androgen-inducedincrease in TGF-β1 could act to suppress lymphocytic infiltration and toattenuate IL-1 and TNF-α production in the lacrimal gland. Thesehormonal effects would then provide a mechanistic explanation for theandrogen-related suppression of autoimmune disease in lacrimal tissueduring Sjogren's syndrome.

As an additional consideration, it is important to note that theandrogen-induced rise in TGF-β content in the lacrimal gland willundoubtedly lead to enhanced secretion of TGF-β by lacrimal tissue andto an increased concentration of TGF-β in tears. Given that TGF-β (e.g.,TGF-β1, TGF-β2) has been detected in tears, measurement of this latterandrogen-associated effect in tears, by utilization of commercial orconventionally-prepared assay kits, would serve as an ideal diagnostictest to monitor the efficacy of topically applied androgens for thetreatment of dry eye.

Androgen Control of the Meibomian Gland

Of particular importance, the potential benefit of topical androgentherapy extends beyond the treatment of aqueous-deficient dry eyesyndromes, as occur during lacrimal gland inflammation in Sjogren'ssyndrome. Topical administration of androgens can also serve as a safeand effective treatment for evaporative dry eye disorders due tomeibomian gland dysfunction.

The meibomian gland is a large sebaceous (i.e., oil-producing) gland(141,142), and androgens are known to control the development,differentiation and function of sebaceous glands in non-ocular sites(141). More specifically, androgens appear to act predominantly onacinar epithelial cells in sebaceous glands, and these cells containboth androgen receptor mRNA and protein (in their nuclei). These acinarcells respond to androgens by increasing both the production andsecretion of lipids. In addition, androgen action in many sebaceousglands is augmented by, or dependent upon, the presence of an enzymecalled 5α-reductase. This enzyme, which has two distinct isozymes (Types1 and 2) encoded by different genes, converts testosterone anddehydroepiandrosterone (native and sulfated forms) into5α-dihydrotestosterone, a very potent metabolite (141,143).

Given this background, it was proposed that androgens may also regulatemeibomian gland function, enhance the quality and quantity of lipidsproduced by this tissue and stimulate the formation of the tear film'slipid layer. In addition, it is proposed that androgen deficiency, asoccurs during menopause (decrease in ovarian androgen and adrenalandrogen precursor secretion), aging in both sexes (decline intesticular androgen output in elderly males), autoimmune disease (e.g.,Sjogren's syndrome, systemic lupus erythematosus, rheumatoid arthritis)and the use of anti-androgen medications (e.g., for prostatichypertrophy or cancer) may lead to meibomian gland dysfunction andconsequent evaporative dry eye.

If androgens do regulate meibomian tissue in a manner analogous to thatof skin sebaceous glands, then meibomian glands should contain androgenreceptor mRNA, androgen receptor protein and 5α-reductase. In addition,androgens should be able to control the synthesis and/or secretion ofmeibomian gland lipids, and androgen deficiency should be linked tomeibomian gland dysfunction and functional dry eye. These hypotheses areshown to be correct in the Example given below.

EXAMPLE VII Effect of Androgens on Meibomian Gland Function

The first set of experiments to test these hypotheses was designed todetermine whether the meibomian gland is an androgen target organ.Tissues were obtained from rats, rabbits and/or humans and analyzed forthe presence of androgen receptor mRNA, androgen receptor protein and5α-reductase mRNA. The results showed that: (a) meibomian glands frommale and female rabbits and humans contain androgen receptor mRNA; (b)meibomian glands of rats and humans contain androgen receptor proteinwithin the nuclei of their acinar epithelial cells; and (c) humanmeibomian glands contain mRNA for both Types 1 and 2 5α-reductase.

The next series of studies demonstrated that androgens influence thelipid profile within the rabbit meibomian gland. Orchiectomized animals(n=5/treatment group) were treated with topical vehicle or19-nortestosterone for 14 days, and meibomian glands were then processedfor lipid evaluation. For comparative purposes, lipid content was alsocharacterized in meibomian tissues of intact, untreated male controls.The results indicated that androgens exert a significant impact on thelipid pattern of the rabbit meibomian gland. Orchiectomy was associatedwith a loss of long-chain fatty acids (FA) in the diglyceride and/ortriglyceride fraction of meibomian gland lipids, whereas the topicaladministration of 19-nortestosterone, but not placebo compounds,restored the FA profile towards that of intact animals. Androgentreatment of orchiectomized rabbits also induced the appearance ofspecific α-hydroxy FA and aliphatic alcohols and increased thepercentage of long-chain FA in the total lipid fraction of meibomianglands.

To determine whether androgen deficiency might be linked to meibomiangland dysfunction and evaporative dry eye, ophthalmic exams wereperformed and meibomian gland secretions were collected from urologicalpatients (n=15) taking anti-androgen medications as well as fromage-matched (n=6) and younger (n=4) controls. The experimental resultsdemonstrated that androgen deficiency appears to be associated withmeibomian gland dysfunction, an altered lipid profile in meibomian glandsecretions, a decreased tear film stability and functional dry eye. Inbrief, the clinical and biochemical results showed that patients, ascompared to controls, had: (a) a higher frequency of light sensitivity,painful eyes and blurred vision; (b) a decrease in the tear film breakup time, a higher frequency of orifice metaplasia, a poorer quality ofmeibomian gland secretions and severe meibomian gland disease; and (c)an attenuation in the amounts of cholesterol esters and wax esters,relative to those of cholesterol, as well as a decreased expression ofspecific molecular species in the diglyceride fraction of meibomiangland secretions.

Overall, these results demonstrate that the meibomian gland is anandrogen target organ, that topical androgens modulate lipid productionwithin this tissue, and that androgen deficiency may cause meibomiangland disease. Moreover, these findings indicate that topical androgenapplication to the ocular surface can serve as a therapy for meibomiangland dysfunction and its associated evaporative dry eye syndromes, notonly in Sjogren's syndrome, but also in other forms of KCS.

USE

Topical application of androgens or their analogues or of TGF-β topatients with KCS or other autoimmune diseases, especially as manifestedin Sjogren's syndrome, can directly suppress the immunopathologicaldefects in accessory lacrimal tissue and the main lacrimal gland'spalpebral lobe, which is adjacent to the ocular surface. Furthermore,topical androgen treatment can increase both the production andsecretion of lipids to reduce meibomian gland dysfunction. Selection ofthe most appropriate therapeutic compounds will depend upon a givenhormone's immunological activity, potential side effects and form ofadministration. For example, topical testosterone may be quite effectivein reducing lacrimal inflammation, and its methylated analogue appearsto have no toxic side effects on parameters such as intraocular pressure(87). However, a variety of other modified and/or anabolic androgens(86,88) may be more effective than testosterone. In addition, withregards to administration, if the androgen is to be complexed to acarrier vehicle (e.g., hyaluronate), then a nitrogenated analogue mightbe indicated.

Therefore, the efficacy of a variety of modified and/or anabolicandrogens in suppressing lacrimal gland autoimmune expression in femaleMRL/lpr mice was compared. Animals were administered vehicle ordesignated androgens systemically for 6 weeks after the onset ofdisease. The androgens examined in this test included: (a) testosterone;(b) dihydrotestosterone (also termed allodihydrotestosterone,androstanolone, stanolone, 5α-dihydrostestosterone); (c)fluoxymesterone; (d) stanozolol; (e) nortestosterone propionate; (f)dehydroepiandrosterone (an androgen precursor, also termedandrostenolone, dehydroisoandro-sterone, DHEA,transdehydroandrosterone); (g) oxandrolone; (h)methyldihydrotestosterone (also termed methylandrostanolone); (i)oxymetholone; (j) 5α-androstan-17β-ol-3-oxime; (k)5α-androstan-17α-ol-3-one-acetate; (1) 2,(5α)-androsten-17β-ol; (m)5α-androstan-2α-methyl-17β-ol-3-one; and (n) methyltestosterone.

The rationale for comparing the immunological activity of this specificarray of androgenic compounds was multifold:

First, these hormones are representative of the major structuralsubclasses of androgens, as disclosed in Vida (88), hereby incorporatedby reference. The subclasses include (a) androgenic compounds withunusual structural features (e.g.,17α-methyl-17β-hydroxy-2-oxa-5α-androstan-3-one, also termedoxandrolone); (b) testosterone derivatives (e.g., methyltestos-terone);(c) 4,5α-dihydrotestosterone derivatives (oxymetholone); (d)17β-hydroxy-5α-androstane derivatives containing a ring A unsaturation,excluding testosterone derivatives (e.g., 2,(5α)-androsten-17β-ol); and(e) 19-nortestosterone derivatives (e.g., 19-nortestosteronepropionate). It may be that certain structural features impart moreoptimal immunosuppressive characteristics, which would be of benefit inselecting specific androgens for human use.

Second, relative to standards (typically testosterone), these androgensinclude compounds displaying: (a) augmented androgenic (i.e.,virilizing) activity coupled with an even larger increase in anabolicactivity (e.g., fluoxymesterone); (b) enhanced anabolic action withunchanged androgenic effects (e.g., oxymetholone, dihydrotestosterone);(c) decreased androgenic ability with unchanged anabolic activity (e.g.,19-nortestosterone propionate); and (d) decreased androgenic capacityparalleled by increased anabolic activity (e.g., oxandrolone,stanozolol). Thus, the analysis should identify an androgen with farmore anabolic, than virilizing, activity to be utilized for thetreatment of ocular manifestations of Sjogren's syndrome (e.g.,oxandrolone possesses 322% of the anabolic and 24% of the androgenicactivity of methyltestosterone (88)). Of course, it is possible thatanabolic effects, per se, may not be involved in androgen suppression oflacrimal autoimmune symptoms. However, the results with19-nortestosterone in MRL/lpr mice demonstrate that this anabolicandrogen, which has significantly reduced androgenic activity inlacrimal tissue (89), was equally as effective as testosterone inabrogating lymphocyte infiltration in the lacrimal gland.

Third, these compounds contain a nitrogen-substituted androgen,5α-androstan-17β-ol 3-oxime, which is created by the substitution of anitrogen derivative for the 3-ketone function in dihydrotestosterone(very potent androgen) (88). This substitution does not inhibit androgenactivity (88) and may permit steroid binding to hyaluronate for topicaladministration. Of interest, a variety of other nitrogenated androgenshave been shown to express increased anabolic, but decreased androgenic,activity. These compounds typically contain 3-substitutions, but notnitrogen incorporation in the steroid ring structure, which appears toabolish androgen action (88).

The results of testing the effect of the representative compounds werethat all androgen classes, whether parental, modified or anabolicanalogue, were effective in suppressing lacrimal gland autoimmuneexpression, although to various degrees. With further routine additionaltesting, the most appropriate therapeutic compound for a specificcondition can be determined. In addition, the therapeutic augmentationof basal tear secretion could allow the use of visual aids, such ascontact lenses, in the Sjogren's syndrome or other autoimmune patientpopulations.

Androgen therapy, which can be administered in the form of drops (e.g.,free hormone, or complexed with carrier substances, such as hyaluronate)or ointment, should not require frequent applications, considering themechanism and duration of androgen/cell interactions. The administrationof a specific compound would be by routine methods in pharmaceuticallyacceptable substances, including buffer solutions (e.g., phosphatebuffered saline) or inert carrier compounds, to the ocular surface oradjacent regions of the eye. Optimal dosage and modes of administrationcan readily be determined by conventional protocols. This treatment can:(a) decrease lymphocyte infiltration in adjacent lacrimal tissue andthereby alleviate immune-mediated destruction, and lymphocytecompression, of acinar and ductal cells; (b) permit accessory and/orpalpebral lacrimal glands to secrete basal tear volumes; it is estimatedthat a tear secretion rate of only 0.1 μl/minute (i.e., one-tenth ofnormal) could maintain a stable tear film under favorable conditions(1); (c) make available regions of functional lacrimal tissue, thatmight respond to exogenous tear stimulants to enhance surface volume;and (d) enhance the function of meibomian glands and thereby promoteincreased stability and decreased evaporation of the tear film.

Topical administration of androgens would avoid the numerous sideeffects of parallel systemic exposure to these hormones, includingvirilization, menstrual irregularities (e.g., amenorrhea), hepaticdysfunction, edema, hematologic abnormalities, behavioral changes andmetabolic alterations. As has been discussed above, androgens arenormally made in the body of all individuals and serve an array ofphysiological functions. In males, the daily production rate oftestosterone equals 2.5 to 11 mg/day, whereas in females it isapproximately 0.25 mg testosterone/day (this amount varies during themenstrual cycle) (86). When androgen replacement therapy is given tocorrect male hypogonadism (i.e., androgen deficiency), the typicalsystemic dosage is 10 to 20 mg androgen/day and is very frequentlydelivered by intramuscular injection; the absolute dosage and specificmethod of administration, though, depend upon the androgen.

For topical androgen therapy, however, the androgen dosage per day wouldbe expected to be far less than the amount normally produced by an adultfemale as seen in the following calculation: Lacrimal glands inexperimental animals contain about 100 fmol cytosol androgen receptor/mgprotein. If an equivalent concentration of receptor occurs in humans,and if one considers the weight of accessory and palpebral lacrimaltissue and level of protein per mg gland weight, then less than 1 ng ofandrogen would be required to saturate all androgen binding sites inhuman lacrimal tissue. This amount is over 250,000-fold less than thedaily testosterone production rate in women.

However, it is understood that the entire dose of androgen would not bedelivered solely to lacrimal tissues; much of the administeredpharmaceutical would be cleared through the nasolacrimal duct.Therefore, an excess over the required dose must be administered. If thedosage were increased 100,000-fold to 0.1 mg, as suggested for thetopical treatment of elevated intraocular pressure (144), then this dosewould still be significantly less than the amount normally produced byadult females. Furthermore, if the topically applied androgen weretestosterone or 19-nortestosterone, an anabolic androgen which hassignificantly reduced androgenic activity in lacrimal tissue asdescribed above, and all of the steroid were to pass through thenasolacrimal canal and to the intestine, then the effective dose wouldbe approximately 0.01 mg (i.e., 10-fold less than the administereddose), due to hepatic metabolism.

To verify that topical androgen administration is inefficient forincreasing serum androgen levels, an experiment was carried out in whichcastrated rabbits were treated for 14 days with either systemic topical19-nortestosterone. The systemic exposure was achieved by theimplantation of subcutaneous, slow release pellets into the subscapularregion. These pellets, which contained 19-nortestosterone were designedto release physiological amounts of androgen over a period of 21 days.The topical exposure involved the application of a drop (40 drops ±oneml) of 19-nortestosterone (1 mg/ml) to both eyes of rabbits twice a day.Systemic androgen treatment was shown to elicit physiologicalconcentrations of serum androgens. In contrast, topical exposure did notraise androgen concentrations in serum: no 19-nortestosterone could bedetected in serum following the topical application of androgens to theocular surface.

TGF-β therapy can be administered in the form of drops or locally byinjection. The administration of a specific compound would be by routinemethods in pharmaceutically acceptable substances including buffersolutions (e.g., phosphate buffered saline) or inert carrier compounds,to the ocular surface or adjacent regions of the eye. The dosage ofTGF-β administered (preferably in the range of 10 pg to 10 mg, and morepreferably 10 ng to 10 μg) can be optimized according to the formulationand method of delivery, and the mode of administration can be readilydetermined by conventional protocols. This TGF-β treatment shouldsuppress lymphocyte infiltration in adjacent lacrimal tissue and therebyameliorate immune-mediated destruction of acinar and ductal epithelialcells.

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While the present invention has been described in conjunction with apreferred embodiment, one of ordinary skill, after reading the foregoingspecification, will be able to effect various changes, substitutions ofequivalents, and other alterations to the compositions and methods setforth herein. It is therefore intended that the protection granted byLetters Patent hereon be limited only by the definitions contained inthe appended claims and equivalents thereof.

What is claimed is:
 1. A method for treating keratoconjunctivitis sicca (KCS) due to androgen deficient disorders and not caused by estrogen deficiency, said method comprisingselecting a patient showing signs or symptoms of aqueous tear deficiency, lipid abnormality, Sjogren's syndrome, lacrimal gland dysfunction, lacrimal gland inflammation or meibomian gland dysfunction; providing a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance; and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of said patient.
 2. The method of claim 1 wherein in said administering step, said therapeutic agent is applied to the ocular surface of the eye.
 3. The method of claim 1 wherein in said administering step, said therapeutic agent is applied to a region of the eye adjacent the ocular surface.
 4. The method of claim 1 wherein in said providing step, said pharmaceutically acceptable substance comprises hyaluronate.
 5. The method of claim 1 wherein in said providing step, said pharmaceutically acceptable substance comprises phosphate buffered saline.
 6. The method of claim 1 wherein said androgen or androgen analogue is from a structural subclass of androgens comprising androgenic compounds with unusual structural features.
 7. The method of claim 6 wherein said androgen or androgen analogue is 17α-methyl-17β-hydroxy-2-oxa-5α-androstan-3-one.
 8. The method of claim 1 wherein said androgen or androgen analogue is a testosterone derivative.
 9. The method of claim 1 wherein said androgen or androgen analogue is a 4,5α-dihydrotestosterone derivative.
 10. The method of claim 1 wherein said androgen or androgen analogue is a 17β-hydroxy-5α-androstane derivative containing a ring A unsaturation.
 11. The method of claim 1 wherein said androgen or androgen analogue is a 19-nortestosterone derivative.
 12. The method of claim 1 wherein said androgen or androgen analogue is a nitrogen-substituted androgen.
 13. The method of claim 1, wherein said patient shows signs or symptoms of aqueous tear deficiency.
 14. The method of claim 1, wherein said patient shows signs or symptoms of lipid abnormality.
 15. The method of claim 1, wherein said patient shows signs or symptoms of Sjogren's syndrome.
 16. The method of claim 1, wherein said patient shows signs or symptoms of lacrimal gland dysfunction.
 17. The method of claim 1, wherein said patient shows signs or symptoms of lacrimal gland inflammation.
 18. The method of claim 1, wherein said patient shows signs or symptoms of meibomian gland dysfunction.
 19. A method for treating keratoconjunctivitis sicca (KCS) due to aqueous tear deficiency, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of aqueous tear deficiency.
 20. A method for treating keratoconjunctivitis sicca (KCS) due to lipid abnormality, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of lipid abnormality.
 21. A method for treating keratoconjunctivitis sicca (KCS) due to Sjogren's syndrome, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of Sjogren's syndrome.
 22. A method for treating keratoconjunctivitis sicca (KCS) due to lacrimal gland dysfunction, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of lacrimal gland dysfunction.
 23. A method for treating keratoconjunctivitis sicca (KCS) due to lacrimal gland inflammation, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of lacrimal gland inflammation.
 24. A method for treating keratoconjunctivitis sicca (KCS) due to meibomian gland dysfunction, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of meibomian gland dysfunction.
 25. A method for treating tear film dysfunction due to aqueous tear deficiency, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of aqueous tear deficiency.
 26. A method for treating treating tear film dysfunction due to lipid abnormality, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of lipid abnormality.
 27. A method for treating treating tear film dysfunction due to Sjogren's syndrome, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of Sjogren's syndrome.
 28. A method for treating treating tear film dysfunction due to lacrimal gland dysfunction, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of lacrimal gland dysfunction.
 29. A method for treating treating tear film dysfunction due to lacrimal gland inflammation, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of lacrimal gland inflammation.
 30. A method for treating treating tear film dysfunction due to meibomian gland dysfunction, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of meibomian gland dysfunction.
 31. A method for treating ocular surface disease due to aqueous tear deficiency, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of aqueous tear deficiency.
 32. A method for treating ocular surface disease due to lipid abnormality, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of lipid abnormality.
 33. A method for treating ocular surface disease due to Sjogren's syndrome, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of Sjogren's syndrome.
 34. A method for treating ocular surface disease due to lacrimal gland dysfunction, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of lacrimal gland dysfunction.
 35. A method for treating ocular surface disease due to lacrimal gland inflammation, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of lacrimal gland inflammation.
 36. A method for treating ocular surface disease due to meibomian gland dysfunction, said method comprisingproviding a therapeutic agent comprising a therapeutically effective amount of an androgen or androgen analogue that has androgenic effectiveness and not estrogen effectiveness in topical application, said androgen or androgen analogue being in a pharmaceutically acceptable substance, and administering said therapeutic agent topically to the ocular surface or immediate vicinity of an eye of a patient showing signs or symptoms of meibomian gland dysfunction.
 37. The method of any one of claims 1 and 13-36 wherein said therapeutic agent is administered topically at a dose rate less than or equal to 0.1 mg/day. 